Since antibodies have high binding activity, binding specificity and high stability in blood, applications thereof to diagnostic, preventive and therapeutic agents for various human diseases have been attempted (Non-patent Reference 1). In addition, human chimeric antibodies or humanized antibodies have been prepared from non-human animal antibodies using gene recombination techniques (Non-patent References 2 to 5). The human chimeric antibody is an antibody in which its variable region is an antibody of non-human animal and its constant region is a human antibody. The humanized antibody is an antibody in which the complementarity determining region (hereinafter referred to as CDR) of a non-human animal is replaced by CDR of a human antibody.
The human chimeric antibodies and humanized antibodies have resolved problems possessed by mouse antibodies and the like, such as the high immunogenicity, low effector function and short blood half-life of non-human animal antibodies, and applications of monoclonal antibodies to pharmaceutical preparations were made possible by using them (Non-patent References 6 to 9). In the Unites States, for example, plurality of humanized antibodies have already been approved as an antibody for cancer treatment, and on the market (Non-patent Reference 10).
These human chimeric antibodies and humanized antibodies actually show effects to a certain degree at clinical level, but therapeutic antibodies having higher effects are in demand. For example, in the case of single administration of Rituxan™ (Non-patent Reference 11) (manufactured by IDEC/Roche/Genentech) which is a human chimeric antibody to CD20, it has been reported that its response ratio for recurrent low malignancy non-Hodgkin lymphoma patients by the phase III clinical test is no more than 48% (complete remission 6%, partial remission 42%), and its average duration of response is 12 months (Non-patent Reference 12). In the case of combination use of Rituxan™ and chemotherapy (CHOP: Cyclophosphamide, Doxorubicin, Vincristine), it has been reported that its response ratio for recurrent low malignancy and follicular non-Hodgkin lymphoma patients by the phase II clinical test is 95% (complete remission 55%, partial remission 45%), but side effects due to CHOP were found (Non-patent Reference 13). In the case of single administration of Herceptin™ (manufactured by Genentech) which is a humanized antibody to HER2, it has been reported that its response ratio for metastatic breast cancer patients by the phase III clinical test is only 15%, and its average duration of response is 9.1 months (Non-patent Reference 14).
The human antibody molecule is also called immunoglobulin (hereinafter referred to as Ig) and classified into respective classes of IgA, IgD, IgE, IgG and IgM based on its molecular structure. The antibody molecule of human IgG (hereinafter referred to as IgG) mainly used as the therapeutic antibody is formed by two respective polypeptides called heavy chain (hereinafter referred to as H chain) and light chain (hereinafter referred to as L chain). The H chain is formed by respective domain structures called H chain variable region (hereinafter referred to as VH), CH1, hinge, CH2 and CH3, from the N-terminal side. The respective domains CH1, hinge, CH2 and CH3 are also called heavy chain constant region as a whole (hereinafter referred to as CH), and the CH2 and CH3 domains are also called Fc region as a whole. The L chain is formed by respective domain structures called L chain variable region (hereinafter referred to as VL) and L chain constant region (hereinafter referred to as CL), from the N-terminal side.
Four subclasses including IgG1, IgG2, IgG3 and IgG4 exist in the IgG antibody H chain. The H chains of respective IgG subclasses mutually have about 95% homology of amino acid sequence in the constant region excluding the hinge which is rich in variability (FIG. 1).
Regardless of the high homology of amino acid sequences in respective IgG subclasses, height of the biological activity possessed thereby varies (Non-patent Reference 15). The biological activity includes effector functions such as complement-dependent cytotoxic activity (hereinafter referred to as CDC), antibody-dependent cell-mediated cytotoxic activity (hereinafter referred to as ADCC) and phagocytotic activity, and these functions play an important role in the living body, such as exclusion of foreign matters and pathogens.
A family of Fcγ receptor (hereinafter referred to as FcγR) are expressed on the surface of various leukocytes such as natural killer cell (hereinafter referred to as NK cell), monocyte, macrophage and granulocyte. The FcγR is classified into active type FcγR including FcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb and suppression type FcγR of FcγRIIb. IgG antibodies, particularly IgG1 and IgG3 in human, strongly bind to these receptors and induce ADCC activity and phagocytotic activity by leukocytes as a result.
The ADCC activity is a cytolytic reaction in which an antibody bound to its antigen binds to mainly FcγRIIIa on the NK cell surface via Fc moiety, and as a result, the reaction is generated by cytotoxic molecules, such as perforin and granzyme, released from the NK cell (Non-patent References 16 and 17). The grade of the ADCC activity is generally in order of IgG1>IgG3>>IgG4≧IgG2 (Non-patent References 18 and 19).
The CDC activity is a reaction in which an antibody bound to its antigen activates reaction cascade of a group of serum proteins, called serum complement system, and finally lyses the target cell. The CDC activity is high in human IgG1 and IgG3, and the grade is generally in order of IgG3≧IgG1>>IgG 2≈IgG4. The complement system is classified into respective components of C1 to C9, and most of them are enzyme precursors which express enzyme activities by partial degradation. The CDC activity starts with the binding of C1q as a component of C1 to the Fc region of an antibody on the target cell, each of the subsequent components is partially degraded by the former step component to advance cascade of the activation, and finally, C5 to C9 form a pore-forming polymer called membrane attacking complex on the cell membrane of the target cell to cause the cell lysis reaction (Non-patent References 16 and 17).
Importance of the above-described effector functions is also recognized on the mechanism of action of therapeutic antibodies used in the clinical field. The above-described Rituxan™ is a human chimeric antibody of IgG1 subclass, and not only it shows ADCC activity and CDC activity in vitro (Non-patent Reference 21) but it has also been suggested on its clinical effects that Rituxan™ actually exerts effector functions in the body of patients, because of the facts that its therapeutic affect is high in the patients showing a genotype of strong ADCC activity (Non-patent Reference 22), that the complement components are quickly consumed from blood after its administration (Non-patent Reference 23), and that expression of CD59 as a factor for suppressing CDC activity increases in cancer cells of relapsed patients after its administration (Non-patent Reference 24). Herceptin™ is also a humanized antibody of IgG1 subclass, and it has been reported that it has ADCC activity in vitro (Non-patent Reference 25).
Based on the above, human IgG1 antibodies are most suitable as therapeutic antibodies, because they have higher ADCC activity and CDC activity and also have longer half-life in human blood than other subclasses.
In order to analyze functions of IgG antibodies, studies have been carried out for the preparation of antibodies in which the domain units were swapped among different IgG subclasses. In the latter half of 1980s, Morrison et al. have pointed out that antibody molecules in which respective domains (CH1, CH2, CH3, hinge) of the heavy chain constant region were swapped between IgG1 and IgG4, or between IgG2 and IgG3, can be expressed as recombinant proteins, and that antibodies in which the hinges of IgG3 and IgG4 were mutually swapped do not show changes in the respective complement fixation capacity and Fc receptor binding ability of the original antibodies (Patent Reference 1). Thereafter, they have examined these domain-swapped antibodies of IgG1 with IgG4 and IgG2 with IgG3 and shown as a result that the C-terminal side of CH2 is important for the CDC activity of IgG1, and CH2 for the CDC activity of IgG3 (Non-patent Reference 26), and that the CH2 domain and hinge are important for the binding of IgG1 and IgG3 to FcγRI which is one of the Fc receptors (Non-patent Reference 27).
It is known that C1q binds to the Fc region of antibody molecules. Binding constants (Ka) of C1q for monomers of human IgG1, IgG2, IgG3 and IgG4 are 1.2×104, 0.64×104, 2.9×104 and 0.44×104 M−1, respectively (Non-patent Reference 20). As described in the above, the CH2 domain is particularly important in the Fc region (Non-patent Reference 26), and more illustratively, it is known that, according to the definition of EU index by Kabat et al. (Non-patent Reference 28), Leu 235 (Non-patent Reference 29) and Asp 270, Lys 322, Pro 329 and Pro 331 (Non-patent Reference 30) in the CH2 are important in the case of human IgG1, and Glu 233, Leu 234, Leu 235 and Gly 236 (Non-patent Reference 31) and Lys 322 (Non-patent Reference 32) in the case of human IgG3.
Attempts have been made to further enhance CDC activity by replacing a part of the amino acid sequence of heavy chain constant region of human IgG3, as a subclass having the highest CDC activity, by an amino acid sequence from other subclass. Regarding hinge lengths of respective IgG subclasses, IgG1 has 15 amino acids, IgG2 has 12 amino acids, IgG3 has 62 amino acids, and IgG4 has 12 amino acids, so that the human IgG3 has a structural characteristic that its hinge region is longer than other IgG subclasses (Non-patent Reference 1). Michaelsen et al. have pointed out that CDC activity of an Ig, in which the 62 amino acids of the hinge of wild type human IgG3 polypeptide were shortened to 15 amino acids by deleting 3 exons of the N-terminal side, exceeds those of IgG3 and IgG1 (Non-patent Reference 33). In addition, Norderhaug et al. have pointed out that the CDC activity is further enhanced when the amino acid sequence of the above-described shortened hinge is allowed to approximate the amino acid sequence of the hinge of IgG4 (Non-patent Reference 34). Also, Brekke et al. have pointed out that in an IgG3 in which its hinge part is replaced by IgG1, and the hinge part, and an IgG3 in which an N-terminal moiety of CH1 is replaced by IgG1, the CDC activity is higher than that of IgG3 and becomes equal to or higher than that of IgG1 (Non-patent Reference 35).
In addition, attempts have also been made to enhance the CDC activity by preparing modified forms of IgG by introducing mutation in all sorts of amino acid sequence in the human IgG heavy chain constant regions, and increasing the binding activity of these modified forms to C1q. Idusogie et al. have reported that the CDC activity is enhanced approximately by 2-fold at the maximum by replacing when Lys at position 326 or Glu at position 333 indicated by the EU index in the CH2 domain in the heavy chain constant region of an anti-CD20 chimeric antibody Rituxan™ having human IgG1 constant region and mouse-derived variable region is replaced by other amino acid (Non-patent Reference 36, Patent Reference 2). Idusogie et al. also have pointed out that the CDC activity of IgG2, which is approximately one-to-several hundreds of the CDC activity of IgG1, increases to about 1/25 of the CDC activity of IgG1, when Lys at position 326 or Glu at position 333 indicated by the EU index is replaced by other amino acid (Patent References 3 to 5).
FcγR-dependent activity such as ADCC activity or phagocytotic activity and CDC activity are both important for the therapeutic effect of therapeutic antibodies. However, since both of the C1q binding as the early stage for inducing CDC activity and the binding to FcγR as the early stage for inducing ADCC activity mediate the antibody Fc, there is a possibility that the ADCC activity is reduced when the CDC activity is enhanced. Idusogie et al. have reported that a point mutation-introduced mutant of Fc amino acids of CDC activity-enhanced IgG shows sharply reduced ADCC activity (Non-patent Reference 36).
Also, it is known that the ADCC activity of an antibody having a human IgG constant region changes by the structure of the complex type N-glycoside-linked sugar chain (its schematic illustration is shown in FIG. 2) to be added to asparagine at position 297 in the CH2 domain (Patent Reference 6). Although there are reports stating that the ADCC activity of antibodies changes depending on the contents of galactose and N-acetylglucosamine in the sugar chain to be bound to the antibody (Non-patent References 37 to 40), the substance which mostly influences on the ADCC activity is a fucose bound to N-acetylglucosamine in the reducing terminal through α1,6 bond. An IgG antibody having a complex type N-glycoside-linked sugar chain in which fucose does not bind to N-acetylglucosamine in the reducing terminal shows remarkably higher ADCC activity than that of an IgG antibody having a complex type N-glycoside-linked sugar chain in which fucose is bound to N-acetylglucosamine in the reducing terminal (Non-patent References 41 and 42, Patent Reference 7). Cells in which the α1,6-fucosyltransferase gene was knocked out are known as the cell which produces an antibody composition having a complex type N-glycoside-linked sugar chain in which fucose is not bound to N-acetylglucosamine in the reducing terminal (Patent References 7 and 8).
Since human IgG3 does not have binding activity to protein A unlike other subclasses (Non-patent Reference 1), it is difficult to purify it when produced as a medicine. It is known that IgG molecules associate with protein A at the interface of CH2 domain and CH3 domain, illustratively, it has been suggested based on an X-ray crystallography that a loop moiety containing amino acids of positions 252 to 254 and positions 308 to 312 indicated by the EU index in the immunoglobulin structure (immunoglobulin fold) of CH2 and positions 433 to 436 in the immunoglobulin structure of CH3 is important (Non-patent Reference 43). It was further shown by a nuclear magnetic resonance method (NMR method) that Ile 253, Ser 254, His 310 and Gln 311 in the CH2 and His 433, His 435 and His 436 in the CH3 of IgG 1 are important (Non-patent Reference 44). In addition, Kim et al. have found that the binding activity to protein A is decreased when His 435 in the human IgG1 heavy chain constant region is replaced with Arg derived from IgG3 (Non-patent Reference 45).    (Non-patent reference 1) Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc. (1995)    (Non-patent reference 2) Nature, 312, 643 (1984)    (Non-patent reference 3) Proc. Natl. Acad. Sci. USA, 81, 6851 (1984)    (Non-patent reference 4) Nature, 321, 522 (1986)    (Non-patent reference 5) Nature, 332, 323 (1988)    (Non-patent reference 6) Immunol. Today, 21, 364 (2000)    (Non-patent reference 7) Immunol. Today, 21, 403 (2000)    (Non-patent reference 8) Ann. Allergy Asthma Immunol., 81, 105 (1998)    (Non-patent reference 9) Nature Biotechnol., 16, 1015 (1998)    (Non-patent reference 10) Nature Reviews Cancer, 1, 119 (2001)    (Non-patent reference 11) Curr. Opin. Oncol., 10, 548 (1998)    (Non-patent reference 12) J. Clin. Oncol., 16, 2825 (1998)    (Non-patent reference 13) J. Clin. Oncol., 17, 268 (1999)    (Non-patent reference 14) J. Clin. Oncol., 17, 2639 (1999)    (Non-patent reference 15) Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc. (1995)    (Non-patent reference 16) Chemical Immunology, 65, 88 (1997)    (Non-patent reference 17) Immunol. Today, 20, 576 (1999)    (Non-patent reference 18) Nature, 332, 323 (1988)    (Non-patent reference 19) Journal of Experimental Medicine, 166, 1351 (1987)    (Non-patent reference 20) Biochemistry, 15, 5175 (1976)    (Non-patent reference 21) Oncogene, 22, 7359 (2003)    (Non-patent reference 22) Blood, 99, 754 (2002)    (Non-patent reference 23) J. Immunol., 172, 3280 (2004)    (Non-patent reference 24) J. Clin. Oncol., 21, 1466 (2003)    (Non-patent reference 25) Cancer Immunol. Immunother., 37, 255 (1993)    (Non-patent reference 26) Journal of Experimental Medicine, 173, 1025 (1991)    (Non-patent reference 27) Journal of Experimental Medicine, 173, 1483 (1991)    (Non-patent reference 28) Sequence of Proteins of Immunological Interest, 5th Edition (1991)    (Non-patent reference 29) Immunology, 86, 319 (1995)    (Non-patent reference 30) J. Immunol., 164, 4178 (2000)    (Non-patent reference 31) Mol. Immunol., 34, 1019 (1997)    (Non-patent reference 32) Mol. Immunol., 37, 995 (2000)    (Non-patent reference 33) Scand. J. Immunol., 32, 517 (1990)    (Non-patent reference 34) Eur. J. Immunol., 21, 2379 (1991)    (Non-patent reference 35) Mol. Immunol., 30, 1419 (1993)    (Non-patent reference 36) J. Immunol., 166, 2571 (2001)    (Non-patent reference 37) Human Antib Hybrid, 5, 143 (1994)    (Non-patent reference 38) Hum Antib Hybrid, 6, 82 (1995)    (Non-patent reference 39) Nat. Biotechnol., 17, 176 (1999)    (Non-patent reference 40) Biotechnol. Bioeng., 74, 288 (2001)    (Non-patent reference 41) J. Biol. Chem., 277, 26733 (2002)    (Non-patent reference 42) J. Biol. Chem., 278, 3466 (2003)    (Non-patent reference 43) Biochemistry, 20, 2361 (1981)    (Non-patent reference 44) FEBS Lett., 328, 49 (1993)    (Non-patent reference 45) Eur. J. Immunol., 29, 2819 (1999)    (Patent reference 1) EP0327378A1    (Patent reference 2) US2003/0158389A1    (Patent reference 3) WO00/42072    (Patent reference 4) US2004/0132101 A1    (Patent reference 5) US2005/0054832 A1    (Patent reference 6) WO00/61739    (Patent reference 7) WO02/31140    (Patent reference 8) WO03/85107